Background: Prenatal ethanol exposure (PNEE) causes irreversible intellectual and behavioral disabilities, clinically known as fetal alcohol spectrum disorder. Few neuropathologic studies of human brain exist. Hypotheses: First, markers of oxidative stress persist following PNEE. Second, PNEE is associated with inhibitory and excitatory neuron changes. Methods: Human brain autopsies (153) with known PNEE were reviewed; 18 cases (fetus to adult) and controls were selected. Oxidative stress and neuronal differentiation markers were used for immunohistochemistry. Results: There were no obvious differences between control and PNEE brains using oxidative stress markers. In human PNEE brains, glutamatergic neurons were reduced 15.96 % and 18.03% in dentate gyrus and temporal cortex, respectively. GABAergic neurons reactive for parvalbumin were reduced in all hippocampal regions (CA1= 57.86%, CA3= 65.15%, and DG= 53.39%) and temporal cortex (44.13%) in all age groups. Conclusion: GABAergic neuron reduction in human following PNEE could explain motor and behavior distractibility in FASD individuals.

Excessive ethanol consumption, characteristic of alcohol use disorders (AUDs), is associated with widespread neurodegeneration and cognitive and behavioral impairments that may contribute to the chronic and relapsing nature of alcoholism. Therefore, identifying novel targets that can afford neuroprotection will undoubtedly aid current treatment strategies for AUDs. The cannabinoids have been shown to provide neuroprotection in a variety of preclinical models of neurodegeneration; however minimal data is available regarding the use of cannabinoid-based pharmacotherapies for treating ethanol-induced neurodegeneration. Therefore, the current dissertation examined the overarching hypothesis: the cannabinoids are a therapeutic strategy to afford neuroprotection in the context of ethanol-induced neurodegeneration. Importantly, this overarching hypothesis was approached with translational considerations in mind. Specifically, the use of many cannabinoids in the clinic is hindered due to multiple unfavorable pharmacokinetic/pharmacodynamic profiles, including high first pass metabolism and untoward psychoactivity. Therefore, the studies herein were designed to circumvent these PK/PD obstacles. The first set of studies examined whether transdermal delivery of the phytocannabinoid, cannabidiol (CBD), could attenuate binge ethanol induced neurodegeneration. Transdermal CBD afforded neuroprotection in the entorhinal cortex and neuroprotection was similar in magnitude as intraperitoneal administration. The second set of studies found that binge ethanol treatment transiently down-regulated the main CNS cannabinoid receptor, CB1R. Interestingly, these changes were not accompanied by alterations in one of the major endogenous ligands, anandamide (AEA), or other related n-acylethanolamides (NAEs). The latter finding is in contrast to other literature reports demonstrating that endocannabinoid content is substantially elevated in response to a CNS insult. Nevertheless, studies were carried out to determine if administration of the AEA and NAE catabolism inhibitor, URB597, could attenuate binge ethanol induced neurodegeneration. URB597 failed to produce neuroprotection in the entorhinal cortex and dentate gyrus of the hippocampus. However, additional studies found that URB597 failed to elevate AEA in the entorhinal cortex, and in general the biological activity of URB597 was impaired by ethanol exposure. Therefore, with further drug discovery/development efforts, it may be feasible to optimize such treatment strategies. In conclusion, the studies within the current dissertation demonstrated the feasibility of using some cannabinoid-based agents to prevent ethanol-induced neurodegeneration.

Chronic ethanol exposure results in neuroadaptations that drive the progression of an alcohol use disorder (AUD). One such driving force is alcohol-induced neurodegeneration. Neuroinflammation has been proposed as a mechanism underlying this damage. Although neuroinflammation is a physiological response to damage, overactivation of its pathways can lead to neurodegeneration. A hallmark indicator of neuroinflammation is microglial activation, but microglial activation is a heterogeneous continuum of phenotypes that can promote or inhibit neuroinflammation. Furthermore acute microglial activation is necessary to restore homeostasis, but prolonged activation can exacerbate damage. The diversity of microglia makes both the level and timecourse of activation vital to understanding their role in damage and/or recovery. The current set of experiments examines the effects of ethanol on microglia within the hippocampus and entorhinal cortex in a binge model of alcohol-induced neurodegeneration. In the first set of experiments, the phenotype of microglia activation was assessed using Raivich’s 5-stages of activation that separates pro- and anti-inflammatory forms of microglia. Morphological and functional assessments suggest that ethanol does not elicit classical microglial activation but instead induces partially activated microglia. In the second set of experiments, the earliest signs of microglial activation were determined to understand the initiation of microglial activation. Experiments indicated that activation occurred subsequent to previous evidence of neuronal damage; however, activation was accompanied by a loss of microglia and the discovery of dystrophic microglia. The final set of experiments examined whether alcohol-induced partial activation of microglia would show a differential response with further alcohol exposure. Experiments showed that animals previously exposed to ethanol showed a greater response to a second ethanol insult. Overall, these studies suggest that although alcohol may initially interrupt the normal microglia response, during abstinence from ethanol a partial activation phenotype appears that may contribute to recovery. Once activated, however, data suggest that these microglia are primed and upon subsequent exposure show an increased response. This heterogeneous microglial response with respect to time does not necessarily reflect a neuroinflammatory response that would be neurodegenerative but does imply that chronic ethanol consumption affects the normal neuroimmune system.

Ethanol exposure during pregnancy is one of the leading causes of preventable birth defects, leading to a range of symptoms collectively known as fetal alcohol spectrum disorder (FASD). More moderate levels of prenatal ethanol exposure (PNE) lead to a range of behavioural deficits including aggression, poor social interaction, poor cognitive performance and increased likelihood of addiction in later life. Current theories suggest that adaptation in the hypothalamic-pituitaryadrenal (HPA) axis and neuroendocrine systems contributes to mood alterations underlying behavioural deficits and vulnerability to addiction. This has led to the suggestion that corticotrophin-releasing factor (CRF) antagonists and glucocorticoid (steroid) inhibitors may be potential therapeutics to address the deficits of PNE and for the treatment of addiction. The zebrafish (Danio rerio) has several advantages over mammalian models, such as low cost of maintenance, short life cycle, easy embryological manipulation and the possibility of large-scale genetic screening. By using this model, our aim is to determine whether developmental ethanol exposure provokes changes in the HPA axis (HPI axis in fish), as it does in mammalian models, therefore opening the possibilities of using zebrafish to elucidate the mechanisms involved, and to test novel therapeutics to alleviate deleterious symptoms. Thus this thesis focuses solely on the effect of developmental ethanol exposure on the functioning of the HPI axis in zebrafish. Stress-reactivity in zebrafish larvae ethanol-treated 1-9 days post 4 fertilisation (dpf) was assessed using thigmotaxis and thigmotaxis following airstress. In both tests, lower stress-related responses were obtained with ethanol treated animals, in that they spent less time at the edges of the apparatus (P<0.01, n=3). They also showed lower total body cortisol (P=0.04, n=14). Larvae also showed the same behaviour pattern two weeks after ethanol exposure, (23dpf) (P=0.04, n=3), again with reduced total cortisol (P=0.03, n=4). HPI-related gene transcription was also assessed in 9dpf ethanol treated zebrafish larvae, by qRT-PCR. Revealing up-regulation of CRH, CRHBP and CRHR2, normalized against β-Actin, Elav1 and Gap43 housekeeping genes. In situ hybridization revealed no spatial changes in CRH, CRH-BP and POMC with animals at the same stage. Behavioural stress-reactivity differences in 6-months old adults that had been exposed developmentally to ethanol were assessed using novel tank diving and thigmotaxis. Both assays indicated a decrease in stress-like behaviour due to early ethanol exposure compared to controls (P<0.05, n=5 both). Finally, cortisol levels were assayed from 9dpf larvae and 6-month-old adults that had been treated with ethanol during early development showed a significant reduction in cortisol output when air-exposed stressed compared to controls (P=0.04, n=5). Conclusion: Early ethanol exposure produced significant changes in cortisol, HPI gene mRNA expression and stress-reactive behaviour in 9dpf animals. Changes in cortisol and behaviour were still detected in 6-months old adults, developmentally treated with ethanol, indicating that early ethanol exposure has permanent effects on the HPI axis. 5 As our data contradicts the findings in mammalian literature where early ethanol exposure increases stress-like behaviour in later life, it is also possible that more permanent effects of PNE in mammals may arise through maternal-offspring interactions, during and post gestation, such as breastfeeding and maternal grooming of the offspring, which are absent in the zebrafish model.

Fetal Alcohol Syndrome (FAS) labels children with physical, mental and behavioral deficits exposed to alcohol in utero. Current research indicates that timing of alcohol exposure of the embryo/fetus is a critical determinant of the behavioral deficits associated with FAS. This study represents a model for binge drinking, in which C57BL mouse embryos were exposed to alcohol during 2 separate critical periods of brain development, gestational day (GD) 7 or 8. As adults, the offspring were tested to determine if loco-motor activity and emotional reaction to a novel environment had been affected. Significant differences due to treatment and sex were noted for both the number of urinations (p=.005 and .001, respectively) and fecal boli (p=.011 and .001, respectively). These results suggest that the quantity of alcohol exposure in utero on the developing brain as in this binge-drinking model is critical in terms of adverse effects on behavioral outcome for the offspring.

Fetal Alcohol Spectrum Disorders (FASD) are the most common cause of cognitive impairment in the United States (Sokol et al., 2003). In young school children in North America and some Western European countries, recent reports have estimated the prevalence of FASD to be as high as 2-5% (May et al., 2009). Currently there are no widely accepted treatment options for FASD, mainly due to the fact that the underlying neurological deficits that occur with prenatal ethanol exposure (PNEE) are still largely unknown. This thesis examines the long-lasting changes that occur in the hippocampus following PNEE using biochemical and electrophysiological techniques. We find that PNEE produces a reduction of the endogenous antioxidant glutathione (GSH), resulting in an increase in oxidative stress that is accompanied by long-lasting reductions in long-term potentiation (LTP) of synaptic efficacy. Interestingly, males exhibited greater deficits in synaptic plasticity than females, despite similar reductions in GSH in both sexes. By depleting GSH in control animals we determined that LTP in the DG of female animals is more resistant to changes in GSH, which may explain the sexual dichotomy observed in these studies of PNEE. Based on these findings, ethanol-exposed animals received postnatal dietary supplementation with either a precursor of GSH, N-Acetylcysteine (NAC) or Omega-3 fatty acids. These supplements helped to counteract the effects of PNEE and improved hippocampal function. The findings in this thesis support the hypothesis that increasing antioxidant capacity can enhance hippocampal function, which in turn may improve learning and memory in FASD, providing a therapeutic avenue for children suffering with these disorders. / Graduate / 0570 Nutrition / 0317 Neuroscience / anna.r.patten@gmail.com

Fetal Alcohol Spectrum Disorders (FASD) are the most common cause of cognitive impairment in the United States (Sokol et al., 2003). In young school children in North America and some Western European countries, recent reports have estimated the prevalence of FASD to be as high as 2-5% (May et al., 2009). Currently there are no widely accepted treatment options for FASD, mainly due to the fact that the underlying neurological deficits that occur with prenatal ethanol exposure (PNEE) are still largely unknown. This thesis examines the long-lasting changes that occur in the hippocampus following PNEE using biochemical and electrophysiological techniques. We find that PNEE produces a reduction of the endogenous antioxidant glutathione (GSH), resulting in an increase in oxidative stress that is accompanied by long-lasting reductions in long-term potentiation (LTP) of synaptic efficacy. Interestingly, males exhibited greater deficits in synaptic plasticity than females, despite similar reductions in GSH in both sexes. By depleting GSH in control animals we determined that LTP in the DG of female animals is more resistant to changes in GSH, which may explain the sexual dichotomy observed in these studies of PNEE. Based on these findings, ethanol-exposed animals received postnatal dietary supplementation with either a precursor of GSH, N-Acetylcysteine (NAC) or Omega-3 fatty acids. These supplements helped to counteract the effects of PNEE and improved hippocampal function. The findings in this thesis support the hypothesis that increasing antioxidant capacity can enhance hippocampal function, which in turn may improve learning and memory in FASD, providing a therapeutic avenue for children suffering with these disorders. / Graduate / 0570 Nutrition / 0317 Neuroscience / anna.r.patten@gmail.com

INTRODUÇÃO: Consumo de álcool na gestação é um sério problema de saúde pública envolvendo grande risco de embriotoxicidade e teratogenicidade fetal. Exposição fetal ao álcool causa liberação de glicocorticóides (GC) pela suprarrenal como conseqüência da ativação do eixo hipotálamo-hipófise-adrenal (HPA). Cortisol é o principal glicocorticóide endógeno capaz de interferir na atividade orgânica, influenciando a retroinibição do eixo HPA. Álcool consumido na gravidez pode alterar indiretamente o desenvolvimento fetal ao perturbar as interações hormonais normais dos eixos hipotálamo-pituitária-adrenal (HPA), hipotálamo-hipófise-tireoidal (HPT), hipotálamo-hipófise-gonadal (HPG), entre mãe e feto. OBJETIVOS: Comparar dosagens de cortisol salivar em crianças em idade escolar, com e sem histórico prévio de exposição intrauterina ao álcool, e sua relação com risco materno para Fetal Alcohol Spectrum Disorder (FASD) e intensidade do uso de álcool na gestação. METODOLOGIA: Amostra foi constituída de 76 pares de crianças e mães, de doze a treze anos de idade. Para análise do cortisol, foi coletada saliva e feitas análises por radioimunoensaio. RESULTADOS: Em relação à caracterização da amostra em função do risco materno se obteve significância para \"mãe praticante de religião\" (X²: 5,60; p=0,01). Associação significativa foi observada entre T-ACE positivo (Tolerance, Annoyed,Cut Down e Eye-Opener) na produção do Cortisol Awaking Response (CAR) e ritmo circadiano em função do sexo da criança (F: 9,26; p=0,003). Diferença significativa foi observada nas análises de níveis de cortisol em função do risco materno para FASD onde as análises de variância (t-tests) do cortisol ao despertar foram encontradas para \"CID positivo\" (Clasificação Internacional de doenças) (t:-2,659; p=0,01) e para cortisol aos 30 minutos depois de despertar em função de uso de álcool na gestação (t: -2,03 ; p=0,05). Em relação aos níveis de cortisol em função do uso de álcool na gestação, se obteve diferenças significativas para o cortisol aos 30 minutos depois de despertar (t: -2,03; p=0,05). Foram observadas diferenças significativas (p<0,01) para seguintes variáveis: níveis de cortisol em função do risco materno para FASD, álcool na gestação versus escore AUDIT (Alcohol Use Disorder Identification Test); Álcool na gestação versus T-ACE; Níveis de cortisol ao despertar versus Níveis de cortisol aos 30 min depois de despertar; Níveis de cortisol aos 30 min depois do despertar versus níveis de cortisol aos 60 min depois de despertar e Níveis de cortisol aos 60 min depois de despertar versus escore do AUDIT. As variáveis, álcool na gestação versus diagnóstico pelo CID, níveis de cortisol ao despertar versus escore do TACE, apresentaram significância (p=0,01). As análises com níveis de cortisol ao despertar versus níveis de cortisol aos 60 min depois de despertar; níveis de cortisol ao despertar versus escore do AUDIT-total; níveis de cortisol aos 30 min depois do despertar versus escore do T-ACE apresentaram significância estatística (respectivamente, p=0,03, p=0,04 e p=0,05). Em relação à avaliação da qualidade do sono em crianças com exposição pré-natal ao álcool por sexo, obteve-se significância para resistência em ir para a cama, para as meninas (p=0,01) e nas análises de correlação se observou diferenças significativas para ansiedade do sono versus níveis de cortisol salivar às 23 horas (p=0,01) e escore do SRQ total versus perturbação respiratória do sono (p=0,02). DISCUSSÃO: Se obteve uma associação entre uso de álcool na gestação e produção de cortisol salivar nos filhos, porém outras variáveis inerentes às mães podem influenciar no desenvolvimento do HPA e na produção de cortisol na pré-adolescência. CONCLUSÃO: Estes resultados podem contribuir para o melhor entendimento da fisiopatologia subjacente às manifestações clínicas de crianças expostas ao álcool durante a gestação e a fundamentar planos de prevenção para evitar que mulheres grávidas consumam álcool na gestação. / INTRODUCTION: Alcohol consumption during pregnancy is a serious public health problem, as it involves great risk related to fetal embryotoxicity and teratogenicity. Fetal alcohol exposure causes the release of glucocorticoids (GC) by the adrenal as consequence of activation of the hypothalamic-pituitary-adrenal (HPA) axis. Cortisol is the major endogenous glucocorticoid able to interfere with the organic activity, influencing retroinhibition of HPA axis. Furthermore, alcohol consumed during pregnancy can alter fetal development indirectly by disrupting the normal hormonal interactions of the hypothalamic-pituitary-adrenal axis (HPA), hypothalamic-pituitarytireoidal (HPT), and hypothalamic-pituitary-gonadal (HPG) between mother and fetus. OBJECTIVES: The objective of this research was to compare the measurements of salivary cortisol in school age children with and without previous history of intrauterine exposure to alcohol, and their relationship to maternal risk for Fetal Alcohol Spectrum Disorder (FASD) and the intensity of alcohol use during pregnancy. METHODOLOGY: The study sample consisted of 76 pairs of children and their mothers, between twelve and thirteen years old. For analysis of cortisol, saliva was collected and analyzes were made by radioimmunoassay method. RESULTS: Results show that, in relation to the characterization of the sample as a function of maternal risk for FASD, significance was obtained for the variable mother religious practice versus score of TACE (Tolerance, Annoyed,Cut Down e Eye-Opener) (X²: 5.60, p=0.01). Statistically significant association was observed between the covariate T-ACE and production of CAR (Cortisol Awaking Response) and circadian rhythm versus sex of the child (F: 9.26, p=0.003). Significant differences were also observed in the analysis of cortisol levels as a function of maternal risk for FASD for the test analysis of variance (t-tests) of cortisol after awakening versus \"negative CID\" and \"positive CID\" (International Clasification of Diseases) (t:-2.659; p=0.01) and cortisol at 30 minutes after awakening versus alcohol use during pregnancy (t:-2.03, p=0.05). In relation to cortisol levels due to the use of alcohol during pregnancy, significant differences were obtained for cortisol at 30 minutes after awakening versus alcohol use during pregnancy (t:-2.03, p=0.05). Significant differences (p<0.01) were found for variables: - cortisol levels as a function of maternal risk for FASD; - alcohol during pregnancy versus score of AUDIT (Alcohol Use Disorder Identification Test); - alcohol in pregnancy versus T-ACE; - cortisol levels after awakening versus cortisol levels at 30 min after awakening; - cortisol levels at 30 min after awakening versus cortisol levels at 60 min after awakening and cortisol levels at 60 min after awakening versus score of AUDIT. Analyses of alcohol during pregnancy versus mother diagnose CID (harmful use or dependence), and cortisol levels after awakening versus score of T-ACE showed significance (p=0.01). The analyses of cortisol levels at 60 min after awakening; cortisol levels after awakening versus AUDIT-total; cortisol levels at 30 min after awakening versus score of T-ACE were significant (respectively, p=0.03, p=0.04 and p=0.05). Regarding the assessment of sleep quality in children with prenatal exposure to alcohol by sex, significance was obtained for resistance to going to bed for female children (p=0.01) and through the analysis of correlation was observed significant results for anxiety sleep versus salivary cortisol levels at 23 hours (p=0.01) and score of mother SRQ total versus respiratory sleep disorder (p=0.02). DISCUSSION: An association was found between alcohol use during pregnancy and salivary cortisol in children of women who consumed alcohol during pregnancy, however other variables inherent to mothers could act in the development of the HPA and the production of cortisol in preadolescence. CONCLUSION: These results can contribute to a better understanding of the pathophysiology underlying the clinical manifestations of children exposed to alcohol during pregnancy and to establish a prevention plan to ensure that pregnant women do not consume alcohol during pregnancy.